EP0977780B1 - Heteropolysaccharid-konjugate, halbinterpenetrierende polysaccharidgele und verfahren zu deren herstellung - Google Patents

Heteropolysaccharid-konjugate, halbinterpenetrierende polysaccharidgele und verfahren zu deren herstellung Download PDF

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Publication number
EP0977780B1
EP0977780B1 EP98920055A EP98920055A EP0977780B1 EP 0977780 B1 EP0977780 B1 EP 0977780B1 EP 98920055 A EP98920055 A EP 98920055A EP 98920055 A EP98920055 A EP 98920055A EP 0977780 B1 EP0977780 B1 EP 0977780B1
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Prior art keywords
alginate
glycosaminoglycan
calcium
biodritin
solution
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French (fr)
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EP0977780A1 (de
EP0977780A4 (de
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Marcos Mares-Guia
Camillo Ricordi
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BIOMM Inc
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BIOMM Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/5073—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals having two or more different coatings optionally including drug-containing subcoatings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/70—Web, sheet or filament bases ; Films; Fibres of the matrix type containing drug
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L17/00—Materials for surgical sutures or for ligaturing blood vessels ; Materials for prostheses or catheters
    • A61L17/14—Post-treatment to improve physical properties
    • A61L17/145—Coating
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0084—Guluromannuronans, e.g. alginic acid, i.e. D-mannuronic acid and D-guluronic acid units linked with alternating alpha- and beta-1,4-glycosidic bonds; Derivatives thereof, e.g. alginates
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
    • C08L5/04—Alginic acid; Derivatives thereof

Definitions

  • This invention relates to methods for producing a novel hetero-polysaccharide conjugate or complex, gels therefrom and/or a composition, e.g., solutions having adjustable viscosity, prepared by manipulating the concentration of hetero-polysaccharide conjugate or complex, and/or ion concentrations, e.g., calcium; and/or a gel or sol comprising the hetero-polysaccharide conjugate; the synthesis, purification and utilization of a novel glycopolymer and/or hetero-polysaccharide and/or gels, solutions and/or sols; and especially, methods for producing a neo-heteropolysaccharide conjugate or complex, herein referred to as "Biodritin heteropolysaccharide", comprising a heteropolysaccharide from covalent bonding between at-least one glycosaminoglycan, e.g., chondroitin sulfate -4 and/or 6, and at least one alginic
  • the invention especially relates to methods for producing Biodritin heteropolysaccharide, gels therefrom and/or a composition, e.g., solutions having adjustable viscosity, prepared by manipulating the concentration of Biodritin heteropolysaccharide, and/or ion concentrations, e.g., calcium; and/or a gel or sol comprising the Biodritin heteropolysaccharide; the synthesis, purification and utilization of Biodritin heteropolysaccharide as a novel glycopolymer and/or heteropolysaccharide and/or gels, solutions and/or sols comprising Biodritin heteropolysaccharide.
  • a composition e.g., solutions having adjustable viscosity, prepared by manipulating the concentration of Biodritin heteropolysaccharide, and/or ion concentrations, e.g., calcium
  • a gel or sol comprising the Biodritin heteropolysaccharide
  • Gels are obtained by adding an inorganic ion, such as calcium ions, to Biodritin heteropolyeaccharide.
  • Sols can be obtained by treatment of a gel with a suitable agent such as a sodium salt, e.g., citrate salts or ethylene-diamine-tetraacetic (EDTA) as sodium salt.
  • Gels can have varying viscosity by varying the amount of Biodritin heteropolysaccharide and/or inorganic ion, e.g., calcium ion; and with an amount of calcium ions, infinite gels can be obtained.
  • the invention further relates to methods for producing a composition herein termed "Biodritin polymer network", and to methods and formulations for preparing a Biodritin polymer network.
  • a Biodritin polymer network comprises at least one glycosaminoglycan, e.g., chondroitin sulfate -4 and/or 6, and at least one alginic acid salt, e.g., sodium alginate, wherein at least one of the glycosaminoglycan and alginic acid salt is cross-linked.
  • the methods and formulations for preparing a Biodritin polymer network comprises admixing the glycosaminoglycan and alginic acid salt and adding at least one cross-linking agent, e.g., an inorganic ion.
  • a Biodritin polymer network which is believed, without necessarily wishing to be bound by any one particular theory, to be a semi-interpenetrating polymer network (s-IPN), is formed by addition of inorganic ions to a solution of a glycosaminoglycan and an alginic acid salt, e.g., calcium ions added to a solution of chondroitin sulfate -4 and/or -6 and sodium alginate (wherein sodium alginate is cross linked and has pockets of chondroitin sulfate -4 and/or -6 and/or non-cross-linked sodium alginate).
  • a glycosaminoglycan e.g., calcium ions added to a solution of chondroitin sulfate -4 and/or -6 and sodium alginate (wherein sodium alginate is cross linked and has pockets of chondroitin sulfate -4 and/or -6 and/or non-cross-linked sodium alginate).
  • a Biodritin polymer network containing at least one Bioditrin heteropolysaccharide can be obtained by covalently bonding the glycosaminoglycan and alginic acid salt present in a Biodritin polymer network, e.g., subjecting a Biodritin polymer network to a coupling reaction involving a linker molecule; for instance, forming network by addition of inorganic ions to a solution of a glycosaminoglycan and an alginic acid salt, e.g., calcium ions added to a solution of chondroitin sulfate -4 and/or -6 and sodium alginate, and subjecting network to coupling reaction, e.g., subjecting network to coupling reaction involving divinyl sulfone.
  • a solution of GAG e.g., chondroitin sulfate-4 and/or -6 is linked to alginate, e.g., sodium alginate by covalent bonding via reaction with a linker molecule such as divinyl sulfone; the reaction is preferably in alkaline medium.
  • GAG e.g., chondroitin sulfate-4 and/or -6
  • alginate e.g., sodium alginate by covalent bonding via reaction with a linker molecule such as divinyl sulfone
  • linker molecule e.g., divinyl sulfone
  • GAG e.g., chondroitin sulfate.
  • Biodritin after the synthesis reaction which involves removal of calcium with molecule which binds to or conjugates with calcium, e.g., EDTA, preferably followed by precipitation(s) with an alchol such as ethanol.
  • the invention also relates to an additional process to prepare a Biodritin polymer network comprising dehydrothermal cross-linking of a mixture of the polymers that form a Biodritin heteropolysaccharide.
  • the dehydrothermal reaction can be of a dry cake containing GAG, e.g., chondroitin sulfate and alginate, e.g., sodium alginate.
  • the cake can be produced by freeze-drying a solution containing chondroitin sulfate and sodium alginate in proper concentrations.
  • the calcium binding sites of alginate can be protected prior to the dehydrothermal treatment by adding calcium ions to bind to the 'egg-box" sites of alginate.
  • the calcium can be removed from the "egg-box" complexes by treatment of the dehydrothermal reaction product with a material which complexes or conjugates or binds with calcium, e.g., sodium EDTA. Purification can be as discussed above.
  • Biodritin polymer networks and Biodritin heteropolysaccharides may be termed “Biodritin” or “Biodritin products”.
  • Biodritin products and/or products derived therefrom is in applications where biocompatibility with host tissues and/or immunoisolation are issues, for example, in cell encapsulation, such as immunoisolation by microencapsulation of islets of Langerhans for diabetes control, or of other cells types or tissues.
  • a Biodritin gel formed from Biodritin heteropolysaccharide or comprised of a Biodritin polymer network e.g., a gel comprising at least one glycosaminoglycan, e.g., chondroitin -4 and/or -6 and at least one alginic acid salt wherein at least one of the glycosaminoglycan and alginic acid salt are cross-linked and the gycosaminoglycan and alginic acid salt are optionally (and preferably) covalently bound, preferably via a reaction involving a linker molecule such as divinyl sulfone, and the cross-linking can be performed after the covalent binding or prior thereto; generally, a Biodritin product or a product from reaction with Biodritin) can be used to form beads or microcapsules containing cells or tissues for implantation. And thus, the invention relates to such beads or microcapsules, and methods for making and using such beads or microcapsule
  • Biodritin products and/or products therefrom can also be "painted", sprayed or applied to or on top of wounds, e.g., surgical wounds or sutures, and can be used to coat surgical, monitoring, or other equipment to avoid local reaction, injury or irritation.
  • wounds e.g., surgical wounds or sutures
  • Biodritin products and/or products therefrom serves as a matrix material for supporting cells for culturing or other applications in which cells must maintain a suspended or non- aggregated state. Accordingly, the invention relates to "paints", sprays, and matrices comprising Biodritin products and/or products therefrom, and to methods for making and using them.
  • novel heteropolysaccharide produced according to the invention and products therefrom, as well as the semi-interpenetrating polymer network, i.e., Biodritin heteropolysaccharide and products therefrom and the Biodritin polymer network make use of: the biocompatibility of glycosaminoglycans (GAGs), preferably of a specific group of glycosaminoglycans, namely those which do not have defined cell binding properties, preferably chondroitin sulfates-4 and/or -6, herein referred to as CIS; and the desirable properties of algal polysaccharides and/or alginic acid,- preferably in the form of a salt of alginic acid, e.g.
  • GAGs glycosaminoglycans
  • CIS chondroitin sulfates-4 and/or -6
  • M is a cation, such as a metal cation, which serves as a stoichiometric counterion to balance negative charges of the alginate anion, e.g., a Group I metal, such as sodium, lithium, or other cations, e.g., organic and complex cations, e.g., ammonium.
  • the counterion is sodium and the salt of alginic acid is sodium alginate, which is able to form infinite gel networks in the presence of calcium ions.
  • Chondroitin sulfates-4 and -6 are preferred as they are able to impart biocompatibility due to the following characteristics: their ubiquity in animal tissues, as components of the extra-cellular matrix or linked to proteins on cell surfaces; their lack of immunogenicity; and their apparent inability to bind specifically to known receptors or cell types. These properties, coupled with covalent bond formation, and the gel-forming properties of salts of alginic acid, forms a basis for the present invention.
  • alginate is used in cell and tissue encapsulation studies, there is considerable discussion as to the extent of its biocompatibility (deVos et al., 1996).
  • the present invention overcomes these obstacles by joining together alginate and CIS, through stable chemical bonds, or in physical gels, e.g., semi-interpenetrating polymer networks, in such a way that the desired properties of each could be preserved and become useful.
  • the invention relates to methods for producing a hetero-polysaccharide, preferably from covalent bonding between at least one glycosaminoglycan, e.g., chondroitin sulfate-4 and/or -6, and at least one alginate or alginic acid salt, e.g., one that forms gels, such as infinite gels in the presence of calcium ions, for instance, sodium alginate, preferably via a coupling reaction with a linking molecule, e.g., DVS.
  • a hetero-polysaccharide preferably from covalent bonding between at least one glycosaminoglycan, e.g., chondroitin sulfate-4 and/or -6, and at least one alginate or alginic acid salt, e.g., one that forms gels, such as infinite gels in the presence of calcium ions, for instance, sodium alginate, preferably via a coupling reaction with a linking molecule, e.g
  • the invention additionally relates to methods for producing a formulation comprising at least one alginate and glycosaminoglycan, e.g., chondroitin sulfate-4 and/or -6 mixed together in solution and polymerized or cross-linked to form a physical gel or polymer network by addition of a polymerizing or cross-linking agent, e.g., calcium ions; e.g, a gel or polymer network which is a semi-interpenetrating polymer network.
  • a polymerizing or cross-linking agent e.g., calcium ions
  • a gel or polymer network which is a semi-interpenetrating polymer network.
  • the invention relates to methods for producing a formulation comprising a gel or polymer network from polymerizing or cross-linking a mixture comprising at least one alginate and at least one glycosaminoglycan; for instance, a formulation comprising a gel or polymer network from adding calcium ions to a mixture comprising at least one alginate and at least one glycosaminoglycan.
  • the invention also relates to methods for producing a formulation comprising a gel from covalently bonding and cross-linking or polymerizing a mixture comprising at least one alginate and at least one glycosaminoglycan; for instance, a gel from adding calcium ions to the inventive heteropolysaccharide, or a gel from subjecting an inventive gel or polymer network to a coupling reaction such as a coupling reaction involving a linker molecule such as DVS.
  • a coupling reaction such as a coupling reaction involving a linker molecule such as DVS.
  • the invention further relates to methods for producing a formulation comprising a combination (mixture) of the inventive heteropolysaccharide and/or gel therefrom and an inventive formulation or a mixture of inventive formulations, e.g., a mixture of inventive gels, described above.
  • the invention even more broadly, relates to:
  • a Biodritin heteropolysaccharide solution is transformed into gel microcapsules by dripping the solution into a calcium chloride solution.
  • a Biodritin heteropolysaccharide solution is fashioned into a slab of any desired shape by contacting the Bioditrin heteropolysaccharide and calcium chloride solution and thus gelling the Biodritin in the forms of the desired shape, such that the shape of the gel may be varied, as desired.
  • the gel microcapsules or slab can have pancreas islets of Langerhans therein.
  • Biodritin can also be formed into a "spaghetti"-like structure, prepared by extrusion of a Biodritin heteropolysaccharide solution over a line, e.g., through a cylindrical catheter tube containing inside a cotton or surgical line.
  • the biodritin solution is extruded together with the line into a calcium ion containing solution, whereby a gel instantly forms that contains the line.
  • the gel is then matured in a calcium ion solution.
  • a protective outer layer e.g., of poly-lysine/Biodritin, over the Biodritin spaghetti, to provide limited permeability to the device.
  • the structuring of the spaghetti device is by the line in the interior and extending further out therefrom towards the surrounding gel cylinder, e.g., by the lateral extensions of the line, or by those provoked on the surface of the line by scraping it, e.g., with a knife blade.
  • a Biodritin spaghetti string can be used for implantation of cells or tissues therein contained, e.g., from the lateral extensions; or more than one Biodritin spaghetti strings may be used by being tied up together at each end.
  • the strings can be further inserted within a biocompatible material such that the biodritin spaghetti strings are protected from mechanical strains after implantation.
  • these string devices can be used as a means of implanting cells or tissues in humans and animals for disease prevention or treatment, as is the case of islets of Langerhans implants for diabetes treatment.
  • composition containing cells such as islet cells
  • cells such as islet cells
  • rate of secretion of a desired expression product of the cells e.g., insulin (see also USSN 08/417,652, filed April 5, 1995, incorporated herein by reference).
  • the invention relates to these products and methods for preparing and using them.
  • Analogous methods can be used to prepare products from Biodritin polymer networks; and, the invention accordingly relates to these products and methods for preparing and using them.
  • glycosaminoglycans such as heparan sulfate, heparin and hyaluronic acid
  • these are not preferred for the invention as they can have defined cell binding properties.
  • hyaluronic acid binds specifically to CD-44, also known as "lymphocyte homing receptor" (Hermes-antigen) or "major hyaluronic acid receptor of mammalian cells” (Aruffo et al, 1990).
  • CD-44 is expressed on the surfaces of most mammalian cells, rodent and primate hematopoietic cell types, fibroblastoid, neural and muscle cells (Rosenmann and St.John, 1993).
  • Hyaluronic acid also binds to Versican, a large proteoglycan secreted by fibroblasts that promotes cell adhesion through interactions with components of the extracellular matrix and cell surface glycoproteins (Zimmermann, 1993).
  • Heparin is also not preferred to be linked to alginate in the composition due to its well known inhibition of the blood clotting mechanism.
  • Heparan sulfate is also not preferred on the basis of its participation in cell-cell adhesion mechanisms. Heparan sulfate is linked to a membrane-bound proteoglycan that binds NCAM (neural cell adhesion molecule), thereby promoting homophilic cell adhesion(Cole et al, 1986).
  • the heparan sulfate binding domain of fibronectin is responsible for the binding of neurons, lymphocytes and other cell types to fibronectin, in the process of cell-cell adhesion (Liao etal, 1988).
  • bFGF basic fibroblast growth factor
  • the inventive heteropolysaccharides made according to the invention arise from stable, covalent chemical bonds between the structures of the at least one alginic acid salt, e.g., sodium alginate and the at least one glycosaminoglycan, e.g., CIS, for example, by a coupling reaction with divinyl sulfone, DVS.
  • these reactants can produce a novel neo-hetero-polysaccharide conjugate having desired properties for use in cell and tissue biology, whenever biocompatibility and/or immunoisolation are critical issues.
  • a semi-interpenetrating polymer network can be prepared by mixing CIS and alginate, at desired concentrations and forming a gel by addition of calcium ions that has similar properties to and benefits of the inventive heteropolysaccharides.
  • the invention can relate at least to two processes.
  • the present invention provides a process for preparing a heteropolysaccharide comprising covalently bonding at least one glycosaminoglycan, e.g., CIS, and at least one alginic acid salt, e.g., sodium alginate, preferably by a coupling reaction with a linker molecule, e.g., DVS, such that the calcium binding sites in alginate are preserved and/or conserved during the coupling reaction.
  • a linker molecule e.g., DVS
  • the present invention relates such products from the heteropolysaccharide and to method for making and using them, e.g, by contacting with ions, such as calcium ions to prepare a gel, for instance, an infinite gel.
  • the second process forms a physical gel, e.g., of the semi-interpenetrating polymer network type, by addition of a polymerizing or cross-linking agent, e.g., calcium ions to a solution containing both at least one alginate and at least one glycosaminoglycan, e.g., chondroitin sulfate-4 and/or -6.
  • a polymerizing or cross-linking agent e.g., calcium ions
  • a solution containing both at least one alginate and at least one glycosaminoglycan e.g., chondroitin sulfate-4 and/or -6.
  • This gel can be a composition that can be varied to suit specific applications.
  • the inventive s-IPN gel is stable and is formed by the complexation of calcium ions with poly-guluronic blocks of alginate to form the "egg-box" structures that stabilize the gel.
  • another formulation can be prepared by suitable combination of the formulations described above, in which a given amount of the covalently bound heteropolysaccharide conjugate is mixed in solution with alginate and CIS, to which solution calcium ions are added to form the gel.
  • another formulation can be prepared by subjecting an inventive gel to a linking reaction so as to covalently bond glycosaminoglycan and alginate units to each other, e.g., by subjecting the gel to a coupling reaction involving a linker molecule such as DVS.
  • a linking reaction so as to covalently bond glycosaminoglycan and alginate units to each other, e.g., by subjecting the gel to a coupling reaction involving a linker molecule such as DVS.
  • the invention further relates to uses of gels and compositions which can be formed into gels, especially biocompatible gels, such as in "paints", sprays, matrices, beads, microcapsules, and the like, including novel uses such as "spaghetti"-like material comprising a spine, e.g., a suture or thread, having a desired material connected thereto, e.g., cells connected to the spine, and an inventive gel surrounding the spine, e.g., in a cylindrical manner.
  • a spine e.g., a suture or thread
  • desired material connected thereto e.g., cells connected to the spine
  • inventive gel surrounding the spine, e.g., in a cylindrical manner.
  • Cells and tissues may be immobilized and immunoisolated by three basic techniques: in extravascular chambers isolated but in the path of the blood stream, in spherical dispersions or microcapsules, and within macrocapsules. Using these techniques a great variety of cells and tissues of different animals have been immunoisolated and implanted in animals for development of therapeutic systems (reviewed by Christenson et al (1993)). Indeed, future applications of immunoisolated cell therapy are envisaged for diseases or conditions such as diabetes, hemophilia, hepatic failure, Alzheimer's, Parkinson's and Huntington's diseases, affective disorders, hepatic failure and fertility problems (Christenson et al, 1993). A complete review of the questions involved in encapsulation and immunoisolation is presented in a recent volume edited by Goosen (1993).
  • insulin-dependent diabetes mellitus In the case of diabetes mellitus, alternatives to daily insulin injections have been searched for control of type I diabetes, or insulin-dependent diabetes mellitus (IDDM). These included pancreas transplants, pumps to deliver insulin under a controlled program and, more recently, Langerhans' islets transplantation. A review of sustained-release implants for insulin delivery has been published (Wang, 1991).
  • GAG's vascular endothelial lining
  • proteoglycans a glycosaminoglycan that is immunogenic; the glycosaminoglycan, e.g., CIS component, is not immunogenic by itself (Hirschmann and Dziewiatkowski, 1966; Loewi and Muir, 1965).
  • a GAG with an alginate salt for instance GAG-alginate biomolecules, e.g., via a coupling reaction with a linker molecule, to form a heteropolysaccharide conjugate and products therefrom and processes and uses of the invention
  • a polymer network e.g., of a semi-interpenetrating polymer network, based on the two components, alginate and GAG, e.g., CIS, rendered in gel form by addition of ions such as inorganic ions, e.g., calcium ions, has not been taught nor suggested in the prior art.
  • a polymer network comprising a heteropolysaccharide from coupling GAG-alginate present in a GAG-alginate polymer network, been heretofore taught or suggested.
  • US Patent No. 4,409,331 to Lim relates to the encapsulation of islets in polymeric material formed from alginate and poly-lysine; Lim and Sun (1980) discussed the microencapsulation of islets to form a bioartificial pancreas.
  • Chitosan microspheres were developed that bind to GAG receptors on cell surfaces (Gallo et al US 5,129,877).
  • Collagen-GAG microcapsules were proposed as drug delivery systems, to deliver anti-microbial agents (Rase et al US 5,169,631).
  • Polyacrylates were also developed as encapsulation materials and have also been co-polymerized with alginate, as discussed by Stevenson and Sefton (1993).
  • GAG-alginate biomolecules e.g., in physical mixture or bound via a coupling reaction with a linker molecule, to form a heteropolysaccharide or a physical network, e.g., s-IPN, gel and products therefrom and processes and uses of the invention, are not taught or suggested.
  • GAG acted as the major biocompatibility agent between a foreign chemical structure or device and host organism, especially in a composition with an alginate.
  • glycoconjugates The synthesis and properties of glycoconjugates has been reviewed in a book edited by Lee and Lee (1994).
  • the new class of structures now being described herein belongs to a group of glycopolymers formed by joining together two different natural heteropolysaccharides by reaction with an unnatural tether e.g., divinyl sulfone. This new class would enter the list of neoglycoconjugates compiled by Magnusson et al(1994) under the heading "glycopolymers with gel forming properties".
  • the physical gel formed by adding calcium ions to solutions containing variable concentrations of GAG, e.g., CIS, and alginate belongs to the group of polymers known as semi-interpenetrating polymer networks, recently discussed by LaPorte (1997). In this category, one type of polymer is enmeshed and entrapped by a second polymer, which is cross-linked to stabilize the structure.
  • compositions of the invention may be characterized as "glycopolymers with gel forming properties" or as a "polymer network”, e.g., "a semi-interpenetrating polymer network” does not mean that the invention has. heretofore been taught or suggested.
  • the present invention provides methods for producing a novel hetero-polysaccharide conjugate or complex, gels and/or sols derived therefrom, and to the synthesis, purification and utilization of a novel glycopolymer and/or heteropolysaccharide and/or gels, solutions and/or sols, herein referred to as biodritin.
  • the hetero-polysaccharide is preferably formed from covalent bonding between at least one glycosaminoglycan, e.g., chondroitin sulfate-4 and/or -6, and at least one alginic acid salt, e.g., such that a gel and/or sol is formed, e.g., in the presence of an appropriate counterion, e.g., calcium.
  • at least one glycosaminoglycan e.g., chondroitin sulfate-4 and/or -6
  • alginic acid salt e.g., such that a gel and/or sol is formed, e.g., in the presence of an appropriate counterion, e.g., calcium.
  • An additional formulation produced according to the invention combines the two polysaccharides, alginate and GAG, e.g., CIS, in aqueous solutions having variable concentrations of either component, through the formation of a gel of the type semi-interpenetrating polymer network, by addition of a cross-linking or polymerizing agent, e.g., calcium ions.
  • a third formulation combines the two concepts in the same preparation, in suitable proportion, which can be established by the skilled artisan, without undue experimentation, from this diclosure and the knowledge in the art.
  • the invention provides methods for producing:
  • the composition can be obtained by admixing 0.5% to 5.0%, e.g., 1% to 3%, by weight or by volume of alginate, e.g., sodium alginate, and about 1.0%, e.g., 1.5% or 2.0%, to up to 20% to 30%, e.g., 25%, by weight or by volume of GAG, e.g., CIS. That is, in general, in forming compositions the alginate to GAG ratio can be 0.5 : 30; but, preferably three (3) to four (4) times more GAG is present than alginate (by weight or by volume), i.e., a preferred alginate to GAG ratio in forming compositions is 1 : 3 to 1 : 4.
  • Alginates come in varying viscosities, e.g., high viscosity, medium viscosity. Medium viscosity alginates are preferred as high viscosity alginates can result in harder beads. Further, the amount of alginate employed in forming an composition can be limited by the viscosity of the alginate; with a medium viscosity alginate, 5% by weight or volume alginate is rather viscous. Furthermore, the skilled artisan can readily appreciate that the volume and viscosity or hardness of compositions, gels and products therefrom, e.g., beads, etc.
  • GAG e.g., CIS
  • alginate e.g., sodium alginate.
  • GAG e.g., CIS
  • alginate e.g., sodium alginate
  • a linker molecule when used in forming an inventive compound, it can be used in an amount relative to the amount of alginate, or to the amount of GAG present, e.g., an amount from 50% of to equal by weight, volume or stoichiometrically to the amount of alginate present, or to the amount of GAG present, or twice or thrice the weight, volume or stoichiometric amount of alginate or GAG present.
  • the inorganic ion is preferably a calcium ion, e.g., calcium chloride, which can be used in an amount relative to the amount of alginate present, e.g., in an amount of about 0.05% to 2.0% by weight or by volume, e.g., about 1.0% by weight or by volume.
  • a calcium ion e.g., calcium chloride
  • Gels can be essentially covalent, or part covalent and part interpenetrating. From analysis of inventive gels, e.g., HPLC analysis showing bands corresponding to alginate, CIS and a covalent structure, it is believed that gels of the invention form interpenetrating networks. However, Applicants do not necessarily wish to be bound by any one particular theory.
  • Biodritin can be applied to surgical wounds or sutures, to protect from adherences; it also serves as a matrix material for supporting cells for culturing or other applications in which cells must maintain a suspended or non-aggregated state.
  • Biodritins are polyanions, carrying both carboxylate and sulfate groups (alginate contains carboxylates, while chondroitin sulfate contains both carboxylates and sulfates); Biodritins form infinite gels when treated with calcium ion, a property imparted by the alginate moiety. Biodritins dissolve readily in water. Additionally, the viscosity of the solutions can be regulated by adjusting the concentration of Biodritin, as well as the concentration of calcium.
  • Biodritins can also be prepared by polymerization of CIS and alginate, in mixed solutions, by addition of calcium ions, thereby resulting in the formation of stable gels of semi-interpenetrating polymer network, in which the CIS component is entrapped in the gel network formed by calcium alginate.
  • Biodritins form stable gels, as the synthesis process of the invention was designed to preserve, with extreme care, the calcium binding sites in the alginate component.
  • the presence of the chondroitin sulfate component imparts a stronger negative charge of sulfates and similarity to an extra-cellular matrix to Biodritins, rendering them entirely biocompatible for applications in transplantation of cells and tissues, among others.
  • Biodritin-derived materials can have their permeability barrier controlled by ionic complexation with positively charged polymers such as poly-L-lysine or poly-L ornitine, which form a thin external capsule to the gel.
  • positively charged polymers such as poly-L-lysine or poly-L ornitine
  • the pore size of this capsule can be adjusted as desired, by changing the concentration and molecular weight of the positively charged polymer during capsule formation.
  • Biodritin preferably has a GAG content ranging from 2 to 200 % of the alginate composition by weight, depending on the specific reaction conditions.
  • the final composition can be adjusted by combining Biodritin that has been cross-linked with Biodritin formed by the gelling of alginate and CIS, in the same solution.
  • the compounds have the desired properties for use as transplantation materials.
  • Insoluble materials can also be prepared by controlling the polymerization conditions. These may have utility as fillers in tissue repair, or as surfaces to allow cell growth in culture.
  • Biodritin beads exposed to an aqueous solvent carries the functionalities of both alginate and chondroitin sulfate (GAG) carboxylates, sulfates, hydroxyls, as well as glycosidic links and the sulfone group of the cross-linker moiety; the negativelly charged sulfate and carboxylate groups are able to form strong ion-ion complexes with polycations such as poly-lysine or poly-ornithine, thereby providing additional external structure and protection to the beads, which has been observed for alginate capsules.
  • GAG chondroitin sulfate
  • the viscous solutions obtained by dissolving Biodritin in water or saline or other desired aqueous medium are easily handled, especially if the concentration is kept under 4 %(w/v) and that they are not exposed to calcium ions.
  • Such solutions can be transformed into infinite gels by complexation with calcium ions; e.g., by dripping them from syringes or capillaries into solutions of calcium chloride one obtains microbeads of desired spherical size.
  • Further treatment with positively charges molecules such as poly-lysine results in a thin external capsule that may, again, be protected by layering Biodritin on top of it from a dilute solution.
  • a Biodritin solution can be extruded, e.g., from a thin tubing directly into a calcium chloride solution, whereby a gel in the form of a rod or cylinder of uniform diameter is obtained. Cells or tissues can be held within these cylinders for the purposes of culturing or transplantation.
  • the biodritin gel may also be conformed, e.g., as a slab of desired size and shape where cells or tissues may become entrapped, for purposes of culturing or implantation.
  • biodritin sol may be "painted” or applied directly with a sterile brush or sprayed around the suture area, followed by spraying with an appropriate calcium chloride solution.
  • a soft, protecting gel forms immediately around the suture area, protecting it from invading cells by blocking cell adhesion.
  • Biodritin gel itself can be sprayed, painted or applied on a suture to protect it from adherences.
  • the skilled artisan can determine the suitable viscosity of the gel and the amount thereof to employ to spray, paint or apply, without undue experimentation, from the knowledge in the art, the disclosure herein, and typical factors such as the wound or suture, and the condition of the patient.
  • the chemical coupling of the two major biodritin components preferably, sodium alginate and chondroitin sulfate-4 and/or -6 is carried out by reaction with a linker molecule, e.g., divinyl sulfone, abbreviated DVS.
  • a linker molecule is a small molecule that is very susceptible to nucleophilic attack (see USSN 08/417,652, with respect to other types of linker molecules and types of coupling reactions).
  • the reaction is carried out at alkaline pH, under conditions which allow the calcium binding sites in alginate to be protected from reaction.
  • the final product of the cross-linking reaction is then rendered soluble and can be separated from the reagents through purification by repeated alcohol precipitation.
  • the concentration of linker molecule, e.g., DVS, used in the reaction can define the final solubility of the glycopolymer complex; beyond a certain range insoluble materials are formed.
  • CIS and alginate An alternative to the chemical coupling of CIS and alginate is the preparation of biodritin by formation of a physical gel from a solution containing both polymers, in desired concentrations.
  • This gel forms when calcium ions are added, and the resulting gel is of the semi-interpenetrating polymer network type, s-IPN.
  • the cross-linked structure is derived from only one component, e.g. alginate, by the formation of the "egg-box" structures resulting from the binding of calcium ions to poly-guluronic blocks in alginate.
  • the resulting gel network entraps and holds the CIS chains that are intertwined with alginate, such that CIS molecules remain as components of the gel structure, imparting to that gel structure the properties of CIS.
  • An element of the invention is the ability of the final product, Biodritin, to form gels with calcium ions. This is achieved by protecting the calcium binding sites in alginate, also called “egg-box” sites, from reacting with the cross-linking reagent during the synthesis of the chemically coupled variant of Biodritin. This is accomplished by adding the appropriate amount of calcium ions to the alginate solution prior to the coupling reaction in such a way that the calcium- "egg-box" complexes are pre-formed, while avoiding strong gelling, and the solution co-polymerization reaction is not blocked.
  • the protecting calcium ions are removed from the "egg-box" complexes, allowing for the dissolution of the particles to form a viscous solution that can, then, be treated as desired.
  • compositions to easily and rapidly go from solution to gel and from gel to solution, by manipulation of the calcium ion concentration, is another significant property of the invention, as it leads to additional applications of this new material outside the transplantation field.
  • the chemical combination of alginate, e.g., sodium alginate and GAG, e.g., chondroitin sulfate-4 and/or -6 is made by dehydrothermal reaction, at temperatures of about 100 °C.
  • DVS is not used and ester bonds are formed between the alcoholic hydroxyls and acidic groups in alginate and chondroitin sulfate.
  • protection of the "egg-box" sites is obtained, and this is useful for preservation of the gelling properties of Biodritin.
  • FIG. 1 A schematic representation of the complex chemical structure of biodritin is shown in Figs. 1 and 4, respectively, for the chemically coupled and the physical gel.
  • Chondroitin sulfate the commercial CIS used contains 70 % of 4-sulfate and 30 % of 6-sulfate; 2.5 g was allowed to dissolve in 25 ml 0.1 M sodium carbonate solution, to give a 100 mg/ml final concentration. In other formulations, higher concentrations of CIS are used, to give final concentrations of 150, 200 or 250 mg/ml.
  • Sodium alginate (5.0 g) was suspended in 45 ml of water, heated to 40 °C, after which another 30 ml water were added in 10 ml portions to facilitate dissolution and decrease viscosity of the final solution.
  • the total blockage of the calcium sites in the alginate solution would require 6.5 ml of a 4.5 M CaCl 2 solution, which is not practical to use.
  • a 4.5 M calcium chloride stock solution was diluted 1: 5 and 200 ⁇ l portions were added at intervals to the sodium alginate solution. During additions, and thereafter, the solution was vigorously mixed with the aid of a power mixer to completely avoid formation of gel clumps. Eight such additions, plus one, were made, in a total of 1800 ⁇ l, corresponding to 1.5 mmol of calcium ions added.
  • Biodritin preparations can also be made in which the calcium binding sites are blocked to a lesser extent, from 25% to 75% of that described above. This also results in soluble materials, provided the coupling with DVS is not extensive.
  • the solution was cooled in an ice-water bath, after which 3 X 100 ml portions of ice-cold ethanol were slowly added with strong mixing with spatula, to precipitate the'product. A white, thread-like, copious precipitate formed; the mixture was left for one hour in the ice-water bath, after which it was centrifuged at 4 °C, at 6000 rpm for a minimum of 20 minutes, in 250 ml plastic bottles.
  • the centrifuge bottle was inverted for drainage of excess liquid, the precipitate was taken and pressed between filter papers, using gloved hands. It was, then, suspended in 50 ml 1 M ethanolamine at pH 9.4, to which 1 ml 0.63 M EDTA was also added. As dissolution started, an additional 50 ml ethanolamine solution was added in two equal portions; small clumps were broken with a spatula. The solution was then stirred while heated to about 40 °C, when complete solution occurred. The container was closed with parafilm and left overnight at room temperature or, alternatively, kept in a water bath at 40 °C for four hours. A golden-yellow, transparent solution resulted that was transferred to a 2 liter beaker for further treatment.
  • the solution of the preceding step was cooled in an ice-water bath and, after 30 min, 3 X 100 ml portions of ice-cold ethanol were added with hefty mixing with spatula. A white, fibrous precipitate formed that sedimented easily by gravity. The suspension was kept for 1 hour in an ice-water bath, after which it was centrifuged at 6000rpm for 20 min at 4 °C, as before. The clear supernatant was discarded and the precipitate collected as in EXAMPLE 4 above and dried between filter papers.
  • This precipitate was re-dissolved in 2 X 50 ml portions of water for a third ethanol precipitation.
  • the solution was transferred to a 2 liter beaker and cooled to ice-water bath temperature. After slow addition of 3 X 100 ml portions of ice-cold ethanol a white, fibrous precipitate formed and the mixture was left for one hour in the ice-water bath.
  • the precipitate was collected after centrifugation carried out exactly as described in this EXAMPLE; it was drained, pressed between filter papers and dissolved, with help of a spatula, in 100 ml water. The final solution was clear, very viscous. It was poured into petri dishes, frozen and freeze-dried.
  • the dry material obtained after freeze-drying was white, weighing, on the average, 5.9 g for this batch size. It was recovered as brilliant, fibrous sheets that break into flakes. The final material is completely soluble in water or 0.15 M NaCl solution. Solutions in both solvents at the levels of 10 to 30 mg/ml form gel spheres when dripped into an 1% calcium chloride solution.
  • Egg-box sites were combined with calcium ions as in Example 2.
  • CIS solution was, then, added to alginate; CIS solution can also be added before the calcium ions addition.
  • a power mixer was used to break any clumps that could have formed at the calcium chloride addition steps. The very viscous final mixture was transferred to petri dishes, frozen and freeze-dried.
  • the hard, dry cake in the petri dishes was then submitted to the dehydrothermal treatment as follows: the dishes containing the cakes were placed in an electric hood maintained at 102-106 °C and kept there for 24 hours. After this time allowed for reaction, the cakes were left to cool to room temperature and further processed.
  • the resulting powder is easily water soluble at concentrations from 10 to 40 mg/ml water or 0.1 M NaCl; the viscosity is a little less than that of equivalent concentrations of Biodritin prepared by chemical co-polymerization with DVS but microcapsules are formed when the solution is dripped into calcium chloride.
  • Biodritin heteropolysaccharide Stock solutions of Biodritin heteropolysaccharide were prepared at 10, 15, 20 mg/ml (DVS polymerized Biodritin) or 30 mg/ml (DHT Biodritin) in 0.15 M NaCl, at room temperature. Dissolution is rapid and easy.
  • the Biodritin heteropolysaccharide solution was dripped from a syringe into a 1.1 % CaCl 2 solution, with mild agitation, from a height of about 15 cm, forming beads. The beads were left to mature for different time intervals in the calcium chloride solution, from 5 to 40 min and were, then, processed as described by Sun et al (1984).
  • microcapsules contained only Biodritin-calcium gel and the outside coatings. Pure microcapsules are used to investigate their biocompatibility in animal experiments.
  • the external diameter of the microcapsules prepared in this EXAMPLE was 4-5 mm but, using devices where a coaxial air stream flows around the syringe needle tip, much smaller microcapsules can be prepared, as is known to those versed in this field.
  • Example solutions were prepared having the following concentrations of components: solvent was 0.15 M sodium chloride; the alginate concentration was fixed at 3.0% and the CIS concentrations used were 1.5%, 3% and 6%, respectively, giving CIS/alginate ratios by weight of 0.5:1, 1:1 and 2:1.
  • the semi-interpenetrating polymer network was prepared in bead form by dropping each of these solutions into a solution of calcium chloride at 1.1%, as described above.
  • Beads were formed from a 20 gauge syringe needle, at a rate of 1 ml/min (peristaltic pump); this needle was inserted into a larger one linked to an air pump so that air could be blown around the dropping needle in order to form microbeads, by fractionation of the out-flowing liquid column, that dropped into a calcium chloride solution.
  • a typical preparation consisted of forming beads from 3 ml of each solution, received in 30 ml 0.15 M NaCl in a small beaker. Immediately thereafter the beads were transferred to a 50 ml centrifuge tube, where they were gently mixed with the calcium solution for 10 minutes. In this step the periods can vary from a few minutes to much longer ones, as 40 min, for example, depending on the extent of gelling that is desired.
  • the beads are centrifuged off at low force (1000rpm, 3 min), transferred to a 15 ml centrifuge tube and washed with 0.15 M NaCl. A washing sequence as described in Example 8 was applied.
  • a poly-lysine capsule can be applied to the beads, exactly as described above.
  • Biodritin beads of the s-IPN type were tested for stability, without the poly-lysine capsule, by incubation for 140 hours at room temperature, in 0.15 M NaCl.
  • Analysis of CIS and alginate leakage made by ultra-violet spectrophotometry at 204 and 215 nm, revealed that only 6% of the total alginate in the beads leaked within the first 50 hours for the beads containing 6% CIS, about 4% alginate leaked from the beads with 3% CIS, and ca. 1% leaked from beads with 1.5% CIS, approximately the same leakage observed with alginate control beads.
  • the leakage of CIS was minimal, in all cases, less than 1%.
  • Example 10 s-IPN gel formed from a mixture of DVS-CIS-crosslinked Biodritin and added CIS
  • a biodritin solution at 2% (w/v) was prepared in 0.15 M NaCl.
  • the biodritin powder preparation used had a DVS-cross-linked CIS content of ca 2% by weight, which was raised to 15% by addition of CIS.
  • the final solution thus, had a composition of 2.0% alginate and 0.3 % CIS, on a w/v basis.
  • Beads were prepared by dropping this solution into 1.1% calcium chloride solution, followed by 40 min incubation in the same solution. Additional treatment included formation of a poly-L-lysine capsule, further layered with biodritin, and washing as described in Example 8.
  • Beads and capsules prepared in this way have identical properties to beads of similar composition prepared from alginate and CIS as in Example 9. They can be treated exactly as the beads described in the next Examples.
  • Biodritin microcapsules can also be sterilized by treatment with 70 % (v/v) isopropanol in 0.15 M NaCl.
  • Biodritin microcapsules were successively treated by Isopropanol/0.15 M NaCl solution of increasing isopropanol concentrations, as follows: 25 %, 50 % and 70 %, all by volume. The first two treatments were applied for 30 min each, to allow slow dehydration of the capsules by the increasing concentrations of isopropanol. When submitted to 70 % isopropanol, the microcapsules were left for up to 4 days at room temperature.
  • microcapsules prepared with alginate alone treated the same way and in parallel experiments, shrunk to 1/3 the original volume and did not recover the initial spherical shape. Most became ellipsoids of revolution, with intact surfaces showing wrinkles that did not disappear with time.
  • Microcapsules treated as in EXAMPLES 8 and 9 were also implanted into mice to test their biocompatibility.
  • Microcapsules were incubated with sodium citrate at room temperature for varying times while observed under a loupe at high magnification. Depending on the capsule size, the time course of events changes but, for a group of larger microcapsules the following results were obtained: after ⁇ 10 min, the central core was surrounded by a narrow, very transparent zone, the latter limited by the external membrane.
  • the membrane Lacking the support of the subjacent gel structure, dissolved by action of citrate, the membrane can be broken by strong aspiration with a pipette.
  • Example 13 Microcapsule Staining with Alcian Blue
  • Alcian Blue is a well known stain for chondroitin sulfate ( Turnbull, 1993); we found that it also stains alginate, although with less intensity. Microcapsules were stained in 0.5 % Alcian Blue in 2% acetic acid, for 20 min; de-staining was in 2 % acetic acid, in repeated washings. Biodritin microcapsules give a deeper blue than alginate capsules, as expected. When Biodritin microcapsules prepared as in EXAMPLE 8, are cut in half and stained, the interior of the capsules stains more intensively than the outside, indicating that the external membrane has an effect on the stain diffusion to the capsule interior.
  • Biodritin spaghetti A new use of the Biodritin invention is in the preparation of structured spaghetti-like cylinders to contain cells or tissues for implantation, itself an invention to be described for the first time herein, called biodritin spaghetti.
  • These Biodritin spaghetti are called "structured” because the thin gel cylinder that receives the name spaghetti has an interior structure formed by a string of sewing cotton line or, alternatively, of surgical suture line.
  • the cotton line has a multitude of very fine lateral extensions, as short branches in a long caulis, that spring from the line surface and are embedded into the gel.
  • the surgical suture does not have such branches, but they can be created by scratching the surface of the line with a knife blade, before sterilizing.
  • These lateral extensions of the central line core provide additional area for adhesion between the gel matrix and the central core, reinforcing the structure in a way similar to the iron wires in concrete.
  • Structured Biodritin spaghetti are prepared as follows: 1) a sterilized cotton sewing line, size 50 or thinner is inserted into a 10 to 20 cm sterile polyethylene catheter of the desired diameter - usually size 60, but size 90 can be used with thinner lines - up to the point where the line reaches the needle, itself connected to a syringe. A surgical suture line can be used in place of the cotton line, with equivalent results. The line should extend beyond the free end of the catheter tubing for 4 to 5 cm, so that in can be handled, anchored or restrained later on in the process (see Figure 2A).
  • solvent saline, culture medium, or Hank's medium
  • a structured spaghetti is formed as follows: the catheter filled with Biodritin heteropolysaccharide solution containing cells and connected to the syringe is quickly placed into a calcium chloride solution in a large petri dish or shallow tray and the syringe plunger is pressed, as the catheter is continuously moved forward in the calcium chloride solution. As the Biodritin heteropolysaccharide solution exits the catheter, it enters into immediate contact with calcium ions and instantly forms a cylindrical gel around the cotton line or surgical line.
  • the Biodritin spaghetti with the line extensions at each end, may be implanted individually or can be tied together by the line ends before inplantation, as shown in Figure 2B. In this way, a much larger mass of cells may be implanted, in a rather limited space, but under conditions that preserve the individual spaghetti.
  • FIG. 7 Another embodiment of the present invention is that a group of Biodritin spaghetti strings with cells, tied together as in Figure 3B, can be placed inside a porous, protective and biocompatible external cylinder initially prepared from veins or arteries of an animal or from the recipient himself. Once attached within such a biocompatible container, to form a sort of living tissue cartridge, the Biodritin spaghetti strings can be implanted into a patient.
  • a possible and interesting alternative proposed in this invention is through the umbelical scar in such a way that, eventually, replacement cartridges may be exchanged using rather simple surgical technique.
  • a Biodritin spaghetti of gel length equal to 10 cm would have the following approximate dimensions ( Figure 4): A) gel length- 100 mm; B)total internal diameter-0.76 mm; C)cotton line diameter- 0.20 mm; D) thickness of Biodritin gel cylinder(B -C)- 0.50 mm; volume of Biodritin gel- 42.2 mm 3 , or 4.22 mm 3 per linear centimeter length.
  • Biodritin microcapsules of 4-5 mm diameter were implanted intra-peritoneally in mice, 3 capsules per mouse, 2 mice per group, following standard surgical procedures and approved protocols.
  • the capsules were removed from the animals.
  • the week-long implanted capsules were found to be free in the peritoneal cavity, clean and with no adherences; no inflammatory reaction could be observed in or around the site where they were found.
  • FIG. 4 shows a capsule removed from a mouse after three months intraperitoneal implantation. Histological study of the preparations fully supported the conclusion: no cells were found on the beads surface after intra-peritoneal implantation for eight days, one month and three months.
  • a 0.8 to 1.5 % (w/v)Biodritin heteropolysaccharide solution in saline is painted on and around a surgical suture; immediately thereafter a 1% (w/v) calcium chloride solution is briefly sprayed as a mist over the painted area. There immediately forms a Biodritin gel over the painted area that protects it from invading cells or from adhering to adjoining tissue. This has important applications in abdominal surgeries, where undesired adherences may form after surgical procedures.
  • the Biodritin heteropolysaccharide solution can be sprayed over and around the suture, followed by spraying of the calcium chloride solution to form the Biodritin gel.
  • a third manner of Biodritin application is to mix the Biodritin heteropolysaccharide solution with an insoluble calcium salt, such as tricalcium citrate, forming an uniform suspension of the calcium salt and, immediately before application, mixing this with an aqueous solution of the ⁇ -lactone of gluconic acid, which slowly hydrolyzes to gluconic acid, which dissolves the calcium ions from the citrate, thereby forming the Biodritin gel in situ .
  • This formulation is called "internal gelation” and has been employed to encapsulate microbial cells by Johansen and Fink (1988).

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Claims (54)

  1. Verfahren zur Herstellung einer biokompatiblen Zusammensetzung, die mindestens ein Glycosaminoglycan und mindestens ein Alginat umfaßt, wobei:
    das mindestens eine Glycosaminoglycan und/oder das Alginat quernetzvernetzt oder polymerisiert ist (sind) und das mindestens eine Glycosaminoglycan und das Alginat kovalent gebunden sind, umfassend die Schritte des Mischens des mindestens einem Glycosaminoglycan und des mindestens einem Alginats und das In-Kontaktbringen des Gemisches mit einem anorganischen Ion und das kovalente Binden des mindestens einem Glycosaminoglycan und des mindestens einem Alginat entweder vor dem In-Kontaktbringen des Gemisches mit dem anorganischen Ion oder nach dem In-Kontaktbringen des Gemisches mit dem anorganischen Ion und Reinigen der Zusammensetzung durch In-Kontaktbringen der Zusammensetzung mit einer Verbindung, die an das anorganische Ion bindet oder es komplexiert oder es konjugiert und das Präzipitieren der Zusammensetzung aus einer Alkohollösung.
  2. Verfahren nach Anspruch 1, wobei das Alginat quervernetzt oder polymerisiert ist.
  3. Verfahren nach Anspruch 1, wobei das anorganische Salz ein Kalziumsalz ist.
  4. Verfahren nach Anspruch 1, wobei das mindestens eine Glycosaminoglycan und das Alginat mittels einer Kopplungsreaktion, die ein Linkermolekül beinhaltet, kovalent gebunden sind.
  5. Verfahren nach Anspruch 4, wobei das Linkermolekül ein Sulfon ist.
  6. Verfahren nach Anspruch 5, wobei das Linkermolekül Divinylsulfon ist.
  7. Verfahren nach Anspruch 1, wobei das mindestens eine Glycosaminoglycan und/oder das Alginat quervernetzt oder polymerisiert ist (sind) und das mindestens eine Glycoaminoglycan und das Alginat kovalent gebunden sind.
  8. Verfahren nach Anspruch 7, wobei das anorganische Salz ein Kalziumsalz ist.
  9. Verfahren nach Anspruch 7, wobei das mindestens eine Glycosaminoglycan und das Alginat mittels einer Kopplungsreaktion, die ein Linkermolekül beinhaltet, kovalent gebunden sind.
  10. Verfahren nach Anspruch 9, wobei das Linkermolekül ein Sulfon ist.
  11. Verfahren nach Anspruch 10, wobei das Sulfon Divinylsulfon ist.
  12. Verfahren nach Anspruch 7, welches dadurch gebildet wird, daß die kovalente Bindung vor dem Quervemetzen oder dem Polymerisieren durchgeführt wird.
  13. Verfahren nach Anspruch 7, welches dadurch gebildet wird, daß die Quervemetzung oder Polymerisierung vor dem Durchführen der kovalenten Bindung durchgeführt wird.
  14. Verfahren nach Anspruch 1, wobei das mindestens eine Glycosaminoglycan keine bestimmten Zellbindungseigenschaften aufweist.
  15. Verfahren nach Anspruch 14, wobei das mindestens eine Glycosaminoglycan Chondroitinsulfat-4 und/oder -6 umfaßt.
  16. Verfahren nach Anspruch 1, wobei das Alginat in der Gegenwart eines anorganischen Ions ein Gel bildet.
  17. Verfahren nach Anspruch 16, wobei das anorganische Ion ein Kalziumion ist.
  18. Verfahren nach Anspruch 17, wobei das Alginat Natriumalginat ist.
  19. Verfahren nach Anspruch 1, wobei das anorganische Salz ein Kalziumion ist, des weiteren umfassend den Schritt des Anpassens der Viskosität durch die Veränderung der Zusammensetzung und/oder der Kalziumionenkonzentration(en).
  20. Verfahren nach Anspruch 1, umfassend den Schritt des Reagierens des mindestens einen Glycosaminoglycan und des Alginats mit einem Linkermolekül und Schützen der Kalziumbindungsstellen des Alginats vor der Reaktion.
  21. Verfahren nach Anspruch 20, wobei das Linkermolekül ein Sulfon ist und die Reaktion im alkalischen Medium durchgeführt wird.
  22. Verfahren nach Anspruch 21, wobei das Sulfon ein Divinylsulfon ist.
  23. Verfahren nach Anspruch 20, des weiteren umfassend das Entfernen der aktiven Vinylgruppen in der Zusammensetzung durch das Reagieren mit einem Alkanolamin bei einem alkalischen pH.
  24. Verfahren nach Anspruch 23, wobei das Alkanolamin Ethanolamin ist.
  25. Verfahren nach Anspruch 1, wobei der Alkohol Ethanol ist.
  26. Verfahren nach Anspruch 1, wobei das anorganische Ion Kalzium ist und die Verbindung EDTA ist.
  27. Verfahren nach Anspruch 1, umfassend das Aussetzen eines trocknen Kuchens, der das mindestens eine Glycosaminoglycan und das mindestens eine Alginat umfaßt, gegenüber einer dehydrothermalen Reaktion.
  28. Verfahren nach Anspruch 27, des weiteren umfassend das Herstellen des trockenen Kuchens durch Gefriertrocknen einer Lösung, die das mindestens eine Glycosaminoglycan und das mindestens eine Alginat umfaßt.
  29. Verfahren nach Anspruch 27, des weiteren umfassend das Schützen der Kalziumbindungsstellen durch das Hinzufügen von Kalziumionen.
  30. Verfahren nach Anspruch 29, des weiteren umfassend die Entfernung von Kalzium aus den Kalziumbindungsstellen durch das In-Kontaktbringen der Zusammensetzung mit einer Verbindung die an Kalzium bindet oder es komplexiert oder es konjugiert.
  31. Verfahren nach Anspruch 30, wobei die Verbindung EDTA ist.
  32. Verfahren nach Anspruch 27, wobei der Alkohol Ethanol ist.
  33. Verfahren für das Zubereiten eines Sols, das das In-Kontaktbringen der Zusammensetzung, die gemäß Anspruch 1 oder 20 hergestellt wurde, mit einem Natriumsalz umfaßt.
  34. Verfahren nach Anspruch 33, wobei das Natriumsalz ein Zitratsalz oder ein Ethylendiamintetraessigsäuresalz ist.
  35. Verfahren für die Zubereitung einer Gelmikrokapsel umfassend das Tropfen einer Lösung, die eine Zusammensetzung, die gemäß dem Verfahren nach Anspruch 1 hergestellt wurde, umfaßt, in eine anorganische Ionenlösung.
  36. Verfahren nach Anspruch 35, wobei die anorganische Ionenlösung eine Kalziumionenlösung ist.
  37. Verfahren nach Anspruch 36, wobei die Kalziumionenlösung eine Kalziumchloridlösung ist.
  38. Verfahren für die Zubereitung eines Gels umfassend das Gestalten der Zusammensetzung, die gemäß dem Verfahren nach Anspruch 1 zubereitet wurde in die Form eines gestalteten Objekts und In-Kontaktbringen der Zusammensetzung mit einem Gelierungsmittel.
  39. Verfahren nach Anspruch 38, wobei das Gelierungsmittel ein Kalziumion umfaßt und die Zusammensetzung in Lösung ist, wenn sie in Form eines gestalteten Objekts gestaltet wird.
  40. Verfahren zur Zubereitung einer Matrix umfassend die Co-Extrusion der Leine, der Zusammensetzung, die gemäß dem Verfahren nach Anspruch 1 zubereitet wurde, und eine anorganische Ionenlösung.
  41. Verfahren nach Anspruch 40, wobei die anorganische Ionenlösung eine Kalziumionenlösung umfaßt.
  42. Verfahren nach Anspruch 41, des weiteren umfassend das Bilden einer äußeren Schicht einer Polylysin-Zusammensetzung über der zylindrischen Struktur.
  43. Eine Matrix die des weiteren innerhalb der Zusammensetzung, die gemäß dem Verfahren nach Anspruch 1 herstellbar ist, Zellen umfaßt.
  44. Matrix nach Anspruch 43, die des weiteren Perlen umfaßt.
  45. Matrix nach Anspruch 43, die des weiteren eine zylindrische Struktur umfaßt.
  46. Matrix nach Anspruch 43, die des weiteren eine Gelmikrokapsel umfaßt.
  47. Matrix nach Anspruch 43 bis 46, wobei die Zellen Langerhans-Inseln der Bauchspeicheldrüse sind.
  48. Verwendung einer Matrix nach Anspruch 47, für die Herstellung eines Medikaments für die Behandlung von Diabetes.
  49. Vorrichtung umfassend mindestens zwei Matrizes nach Anspruch 45, die an jedem Ende zusammengebunden sind.
  50. Vorrichtung nach Anspruch 49, die des weiteren ein zusätzliches biokompatibles Material umfaßt, das die zylindrische Struktur zum Schutz gegen mechanische Belastungen umgibt.
  51. Vorrichtung nach Anspruch 49, wobei die Zellen Langerhans-Inseln der Bauchspeicheldrüse sind.
  52. Verwendung einer Vorrichtung nach Anspruch 51, für die Herstellung eines Medikaments für die Behandlung von Diabetes.
  53. Verwendung einer Farbe oder eines Sprays umfassend eine Zusammensetzung, die gemäß Anspruch 1 hergestellt wurde, für die Herstellung eines Medikaments für die Behandlung von Wunden oder die Abdeckung von chirurgischen Nähten.
  54. Verfahren für das Abdecken von Operationsgeräten umfassend das Anbringen einer Farbe oder eines Sprays umfassend eine Zusammensetzung, die gemäß Anspruch 1 zubereitet wurde.
EP98920055A 1997-04-30 1998-04-30 Heteropolysaccharid-konjugate, halbinterpenetrierende polysaccharidgele und verfahren zu deren herstellung Expired - Lifetime EP0977780B1 (de)

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US4511197P 1997-04-30 1997-04-30
US45111P 1997-04-30
US877682 1997-06-17
US08/877,682 US6281341B1 (en) 1997-04-30 1997-06-17 Hetero-polysaccharide conjugate and methods of making and using the same
PCT/US1998/008752 WO1998049202A1 (en) 1997-04-30 1998-04-30 NOVEL HETERO-POLYSACCHARIDE CONJUGATES, NOVEL s-INP POLYSACCHARIDE GELS AND METHODS OF MAKING AND USING THE SAME

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CA2597757C (en) 2005-02-10 2016-06-28 Regents Of The University Of Minnesota Vascular/lymphatic endothelial cells
FR2925316B1 (fr) * 2007-12-21 2010-02-12 Oreal Kit comprenant un alginate et un agent de complexation sous forme de sel insoluble dans l'eau
FR2925310B1 (fr) * 2007-12-21 2010-03-05 Oreal Kit de mascara comprenant un alginate et un agent de complexation
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FR2925315B1 (fr) * 2007-12-21 2010-02-12 Oreal Kit comprenant un alginate et un agent de complexation sous forme de sel hydrosoluble
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WO2011160066A1 (en) 2010-06-17 2011-12-22 Regents Of The University Of Minnesota Production of insulin producing cells
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BR9809326A (pt) 2000-07-04
WO1998049202A1 (en) 1998-11-05
ATE275585T1 (de) 2004-09-15
EP0977780A1 (de) 2000-02-09
US6281341B1 (en) 2001-08-28
DE69826119T2 (de) 2005-09-15
EP0977780A4 (de) 2002-02-27

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